Suppression of lymphocyte proliferation and regulation of dendritic cell phenotype by soluble mediators from rat lacrimal epithelial cells.

Lacrimal epithelial cells appear to constitutively secrete autoantigens to their underling stroma. The present experiments address the hypothesis that they also secrete soluble factors that regulate immune responses. Epithelial cells, spleen cells and lymphocytes were obtained from rabbits or rats and cultured in various configurations. Monocytes from rat bone marrow were matured to dendritic cells (DC) ex vivo. Proliferation was measured by [3H]-thymidine incorporation; surface MHC Class II and CD86 using flow cytometry; and mRNA relative abundances using real time RT-PCR. Microporous culture inserts containing rat lacrimal cells inhibited proliferation of rabbit lymphocytes co-cultured with autologous lacrimal cells and of rat lymphocytes co-cultured with TNF-alpha-stimulated DC. They inhibited CD86 and MHC Class II surface expression by maturating DC and reversed surface expression of CD86 but not MHC Class II by partially matured DC. Subsequent exposure of partially matured DC to mediators from rat lacrimal cells reversed the ability to stimulate lymphocyte proliferation. TGF-beta(1) and IL-10 mRNAs increased somewhat when rat lacrimal cells were isolated but decreased markedly in rabbit lacrimal cells. Antibodies to TGF-beta prevented soluble factors from rat lacrimal cells from inhibiting proliferation of rabbit lymphocytes co-cultured with rabbit lacrimal cells, but recombinant TGF-beta alone did not mimic the soluble factors. IL-10 immunopositivity was detected in epithelial cells of interlobular ducts and occasional interstitial cells in rabbit lacrimal gland. Rat lacrimal epithelial cells secrete TGF-beta and other factors that synergize to suppress lymphocyte proliferation and regulate DC maturation. Interlobular duct epithelial cells in rabbit lacrimal glands may express similar functions.

Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium.

This study reports on the distribution of bicarbonate-stimulated ATPase in rat intestinal epithelial cells. Brush-border membranes and basolateral membranes were separated from each other and from mitochondrial and other intracellular membranes by differential and density gradient centrifugation. Bicarbonate-sensitive ATPase activity followed the mitochondrial marker succinic dehydrogenase closely throughout all the centrifugation steps. The low HCO3--ATPase activity in purified brush-border and basolateral plasma membranes could be accounted for quantitatively by the small mitochondrial contamination. Consequently, there are no grounds for postulating that this enzyme has a direct role in H+ or HCO3- transport across the rat small intestine.

Subcellular distribution of ATP-dependent calcium transport in rat duodenal epithelium.

Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome -c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker (Na+-K+)-ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter-current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of (Na+-K+)-ATPase, contained ATP-dependent Ca2+-transport activity with Vmax = 3.7 nmol/mg per min and Km = 0.5 microM. A major Golgi-derived population exhibited Ca2+-transport activity with Vmax and Km values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca2+-transport activity with Vmax = 4 nmol/mg per min and Km = 0.5 microM. In addition to demonstrating that ATP-dependent Ca2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca2+-pumping ability.

Sugar uptake by intestinal basolateral membrane vesicles.

A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells.

Apical and basal-lateral Na/K-ATPase in rat lacrimal gland acinar cells.

The distribution of Na/K-ATPase in rat exorbital lacrimal gland was studied using immunofluorescent localization of an antibody raised against rat kidney Na/K-ATPase. In cryostat sections, intralobular ducts were strongly immunoreactive and acinar cells showed localization on both apical and basal-lateral surfaces. Acinar cells also had strong intracellular reactivity in apical regions. These results are discussed in light of current models of exocrine gland electrolyte secretion.

Traffic of major histocompatibility complex class II molecules in rabbit lacrimal gland acinar cells.

PURPOSE: It has been suggested that lacrimal gland acinar cells, which have been induced to express major histocompatibility complex class II (MHC II) molecules, might initiate local autoimmunity by using mechanisms similar to those operating in the specialized antigen-presenting cells to process and present autoantigens. Surface-labeling experiments indicate that constituents of the acinar cell plasma membrane participate in a rapid recycling traffic. The authors have surveyed the subcellular distribution of MHC II molecules and have evaluated their participation in the traffic between plasma membranes and intracellular compartments. METHODS: Acinar cells were isolated from rabbit lacrimal glands and maintained for two nights in a serum-free, hormone-supplemented culture medium containing 10 microM carbachol. MHC II molecules were detected with a monoclonal antibody (MAB 2C4), biotinylated goat-antimouse IgG (BGAM), and avidin-ferritin (AvFe) or streptavidin-gold (SAvAu) conjugates. RESULTS: Postembedding labeling with MAB 2C4, BGAM, and AvFe revealed MHC II molecules at the surface membranes, in cytoplasmic vesicles, and in secretory vesicles. When cells were chilled to 4 degrees C and subjected to preembedding labeling with MAB 2C4, BGAM, and AvFe, surface MHC II molecules were specifically labeled. Labeled complexes were rapidly internalized upon warming to 37 degrees C. Postembedding labeling with MAB 2C4, BGAM, and SAvAu revealed additional intracellular MHC II molecules, with a distribution overlapping that of the MHC II molecules labeled during the preembedding procedure. When cells were cultured overnight in the presence of MAB 2C4 and subjected to postembedding labeling with BGAM and AvFe, label was detectable in small vesicles and in secretory vesicles. However, the extent of labeling appeared less than obtained with postembedding labeling with MAB 2C4, BGAM, and AvFe. Preembedding labeling of cells that had been incubated overnight with MAB 2C4 indicated that the cells continued to express MHC II molecules at their surface membranes, and rapid internalization of label upon warming to 37 degrees C confirmed that MHC II molecule traffic continued in the presence of MAB 2C4. Postembedding labeling with MAB 2C4, BGAM, and SAvAu indicated the continued presence of a large intracellular pool of MHC II molecules. CONCLUSIONS: MHC II molecules in lacrimal acinar cells are present in large intracellular and small surface pools. They move rapidly between these two pools, but further work will be required to determine whether the MHC II molecule traffic represents recycling or turnover and whether recycling pools and sequestered pools coexist. The presently available data make it reasonable to propose that the traffic of MHC II molecules to plasma membranes provides a mechanism by which acinar cells display intracellularly generated autoantigens to potentially reactive helper T lymphocytes.

Analytic subcellular fractionation of acini from rat lacrimal gland.

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.

Plasma membrane internalization and recycling in rabbit lacrimal acinar cells.

PURPOSE: The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells. METHODS: Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4 degrees C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidin-lucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with avidin-ferritin conjugate for electron microscopy. RESULTS: At 4 degrees C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37 degrees C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in multivesicular bodies. Incubation at 37 degrees C before chilling and labeling with avidin-lucifer yellow decreased the cells' capacity to bind avidin-lucifer yellow by 95%, with t0.5 < 0.5 min. If cells were then incubated with avidin-lucifer yellow at 37 degrees C, they took up the marker with a time course that indicated that 60% of the initial biotin either recycled back to the plasma membrane or remained in intracellular compartments that could be reached by endocytosed extracellular fluid. Internalized biotin communicated with extracellular avidin-lucifer yellow with a t0.5 of 2 min, and this process was accelerated by carbachol at concentrations of 10 mumol/l and 1 mmol/l. CONCLUSIONS: Acinar cell plasma membrane constituents participate in an ongoing, secretagogue-modulated recycling traffic between small surface-expressed pools and 10- to 20-fold larger intracellular pools.

Rabbit lacrimal acinar cells in primary culture: morphology and acute responses to cholinergic stimulation.

PURPOSE: The rabbit lacrimal gland yields large numbers of viable acinar cells that, when exposed to carbachol, respond with accelerated protein release, fluid phase endocytosis (Lucifer yellow uptake), and Na/H antiport activation. The current study was undertaken to determine whether such cells exhibit similar responses after having been maintained in primary culture. METHODS: Cells were isolated from 2-kg, juvenile male New Zealand White rabbits and maintained in a supplemented DMEM/Ham's F-12 medium for up to 72 hours. RESULTS: Electron microscopy showed the organization of freshly isolated cells to be highly polarized, with secretory vesicles at one pole and nucleus at the other; vesicles were heterogeneous in size and in the electron density of their contents. The cells remained polarized after overnight culture, but the secretory vesicle population was more homogeneous in size and content, and the cells tended to aggregate. After 72 hours, roughly half the cells retained good morphology and cytoplasmic polarization, but the vesicles were enlarged and their contents less electron dense. Cells that had been maintained overnight responded to the addition of 10 microM carbachol with a 32.2% +/- 15.5% (n = 12, P < 0.04) increase in the total amount protein released during a standard 20-minute incubation. This represented a mean 125% increase in the temperature-dependent component of protein release. The protein secretory response was decreased to 14.6% +/- 6.1% (n = 3, P < 0.07) for cells that had been maintained for 72 hours. In the same samples, carbachol increased fluid phase endocytosis by 38.3% +/- 8.1% (P < 0.01) and 70.9% +/- 13.4% (P < 0.025), respectively. The protein secretory response was partially, and the endocytic response fully blocked by 1 mM atropine. CONCLUSIONS: This model could be useful as a simplified system in which to study regulation of acinar cell function over days, rather than hours, as is required in fresh tissue models.

Subcellular distribution of muscarinic acetylcholine receptors in rat exorbital lacrimal gland.

The muscarinic acetylcholine receptor (MAChR) is an important mediator of parasympathetic regulation of secretion by the rat exorbital lacrimal gland. In order to survey the subcellular distribution of MAChR in lacrimal acinar cells, we have measured the binding of the specific muscarinic cholinergic antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB) to membrane samples isolated from rat exorbital lacrimal glands by differential and equilibrium density gradient centrifugation. Binding of [3H]-QNB in all membrane fractions was consistent with the presence of a single class of receptor which was muscarinic in nature on the basis of its Kd for [3H]-QNB (0.30-0.35 nM) and its ability to interact with the muscarinic agonists carbachol and methachol and the antagonist atropine. MAChR were present at the highest specific activity in acinar cell basal-lateral plasma membrane-derived populations, where Bmax was as high as 1960 fmole/mg protein. However, the density distributions of MAChR and of other membrane markers indicated that the receptors were present also in membranes derived from cytoplasmic structures, where Bmax values ranged from 50.4 to 152.8 fmole/mg protein. Stimulation with 10 microM carbachol for 30 min led to a 20% (P less than 0.05) increase in the relative MAChR content of a population of membranes derived from the acinar cell basal-lateral membrane; an apparent tendency for MAChR activity to decrease in other membrane populations suggests that stimulation might cause a redistribution of MAChR between cytoplasmic pools and the cell surface membranes.

Prolactin localization, binding, and effects on peroxidase release in rat exorbital lacrimal gland.

Prolactin immunoreactivity has been detected in human tears and in lacrimal glands, and it has been suggested that this hormone might be a modulator of lacrimal secretion as well as a component of lacrimal gland fluid. The present study was designed to confirm the immunocytochemical localization of prolactin in the rat lacrimal gland, to determine the source of the prolactin, and to evaluate the acute effects of prolactin on lacrimal secretory function. We have confirmed that prolactin-like immunoreactivity is present in secretory vesicles of acinar cells of male and female Sprague-Dawley rats. Prolactin message was present at detectable levels in RNA extracts of lacrimal glands from males, indicating that at least a component of the prolactin-like immunoreactivity was the product of synthesis within the lacrimal glands. Crude membrane fractions from acini isolated from males bound 43.1 +/- 3.2 femtomoles prolactin/mg protein (mean +/- standard error of the mean; n = 6), which was significantly (P less than 0.01) more than comparable fractions from females (15.4 +/- 2.4 fmoles/mg protein, n = 6). Preincubating membranes at 65 degrees for 20 min to release endogenous ligands increased prolactin binding to 84.8 +/- 20.8 fmoles/mg protein for males and 63.8 +/- 17.4 fmoles/mg protein for females (P greater than 0.1), suggesting that, on average, similar numbers of receptors are expressed in acinar cells of male and female rats but a larger fraction of the receptors is occupied by endogenous prolactin-like peptides in females. Because prolactin binding triggers prolactin receptor internalization in various cell types, we propose that the prolactin-like immunoreactivity in lacrimal acinar cells of females has been accumulated from the circulation, while the immunoreactivity seen in males results, at least in part, from de novo synthesis. Ovine prolactin at concentrations of 10-20 ng/ml inhibited carbachol-induced peroxidase release by 19.6% +/- 6.9% (n = 8, P less than 0.02) but failed to alter peroxidase release in the absence of carbachol. These observations suggest that prolactin might function as an endocrine, paracrine, or autocrine modulator in the lacrimal gland.

Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini.

Lacrimal acinar cells secrete macromolecular products in an approximately isotonic, sodium chloride (NaCl)-rich fluid. The mechanisms of macromolecular product secretion depend in part on a recycling traffic of membrane constituents between the Golgi complex and the apical plasma membrane. In contrast, the acinar cell's mechanisms for secreting Na+ and Cl- depend largely on the fluxes of these ions through transporters expressed in the apical and basal-lateral membranes. In addition to accelerating the recycling of secretory vesicle membrane constituents, the cholinergic agonist carbachol also triggers a net redistribution of sodium potassium adenosine triphosphatase (Na,K-ATPase) ion pumps between Golgi-associated pools and the basal-lateral plasma membranes (Yiu SC, et al: J Membrane Biol 102:185, 1988). In the present study, acinar preparations from rat lacrimal glands were stimulated with either carbachol, epinephrine, or isoproterenol. All three agonist stimulated release of the secretory protein lactoperoxidase, but only carbachol significantly accelerated Na+ undirectional influx. Subcellular fractionation analyses of resting and stimulated preparations indicated that carbachol caused a significant translocation of Na,K-ATPase activity from a Golgi-associated compartment to the basal-lateral plasma membranes. Neither adrenergic agonist significantly increased the basal-lateral membrane Na,K-ATPase activity, but each triggered a distinct pattern of redistributions of Na,K-ATPase and the Golgi membrane marker, galactosyltransferase. The carbachol-induced augmentation of basal-lateral membrane Na,K-ATPase activity represents a mechanism by which the cell might compensate for increased Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.

The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.

Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells.

It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is involved in T cell activation and leads to autoimmunity. Although Class II antigen expression was found in various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells has not been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for the numbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages. None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosis characteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases; they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases, there were greater numbers of T cells per millimeter squared (229 +/- 94 [mean +/- the standard error of the mean]) than in the two HLA-DR-negative cases (37 +/- 1 [mean +/- range]). Three lacrimal gland specimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three specimens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands and cultured overnight in serum-free media supplemented with several potential mediators of Class II antigen expression: interferon-gamma, carbachol, or isoproterenol. Freshly isolated cells did not express Class II antigens at detectable levels, but in most experiments, they began to express the antigen even in the absence of putative mediators.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium.

We have attempted to isolate samples of apical and basal-lateral plasma membranes from cultured fetal human RPE. Cells from confluent, dome-forming cultures were disrupted with a Dounce apparatus. Nuclei and melanin granules were sedimented by centrifugation at 2600 g for 10 min. The supernates were layered over gradients of 17.5-65% sorbitol and centrifuged at 122,000 g for 5 hr. Fractions were grouped into "density windows" on the basis of their biochemical marker contents. Na,K-ATPase and alkaline phosphatase overlapped but did not precisely parallel one another, suggesting associations with two partially separated membrane populations; in density window I, alkaline phosphatase was enriched 4.3-fold, and Na,K-ATPase was enriched 1.7-fold, whereas in window II the corresponding enrichment factors were 7.7 and 6.7. These markers were well resolved from a mitochondrial marker, but they were overlapped by endoplasmic reticulum and Golgi markers. Additional density gradient centrifugations, performed after samples had been suspended in 55% sorbitol, further separated alkaline phosphatase- and Na,K-ATPase-containing membranes from endoplasmic reticulum and Golgi membranes, yielding alkaline phosphatase and Na,K-ATPase cumulative enrichment factors of 6.8 and 2.5 for the sample from window I and 9.3 and 10.9 for the sample from window II. Subsequent phase partitioning analysis of the sample from window I further enriched an alkaline-phosphatase-rich membrane population, which is believed to represent the RPE basal-lateral membranes. The sample from density window II contained two membrane populations, both enriched in Na,K-ATPase, alkaline phosphatase, and galactosyltransferase, and both of which appear to be derived from the apical plasma membrane. SDS-PAGE and Western blotting confirmed a correlation between Na,K-ATPase catalytic activity and Na,K-ATPase alpha subunit immunoreactivity.

Na+/H+ antiporter in lacrimal acinar cell basal-lateral membranes.

The first step in the formation of lacrimal gland fluid is believed to depend on transport systems which couple a flux of Cl- ions to the passive influx of Na+ ions across the acinar cell basal-lateral plasma membrane. The transport systems which mediate these fluxes have not yet been characterized, but a review of previous studies (Parod and Putney, Am J Physiol 239:G106, 1980) raises the possibility that Na+/H+ antiporters might represent a major pathway for Na+ influx. This conclusion is of interest, because antiporter mediated Na+ fluxes can, potentially, drive net Cl- fluxes. We have now examined a sample of basal-lateral membrane vesicles from rat exorbital lacrimal gland to verify the presence of a Na+/H+ antiporter activity. Imposition of an outward H+ gradient caused a 4.4-fold increase in the 22Na influx rate, while imposition of an outward Na+ gradient accelerated H+ uptake as determined by changes in acridine orange absorbance. All transport experiments were done in the presence of valinomycin and symmetrical K+ concentrations, eliminating the possibility of conductive Na+ or H+ fluxes driven by diffusion potentials. The pH gradient dependent Na+ influx was completely inhibited by 1 mM amiloride, indicating that it was mediated by a Na+/H+ antiporter similar to those described in other tissues. Comparison of the density distributions of Na+/H+ antiport and standard membrane marker enzyme activities confirmed that the antiporter was primarily localized to the basal-lateral membranes.

Hypophysectomy-induced regression of female rat lacrimal glands: partial restoration and maintenance by dihydrotestosterone and prolactin.

PURPOSE: Previous studies have implicated androgens and one or more as yet unknown pituitary or pituitary-dependent factors in the regulation of certain lacrimal gland functions. Many observations suggest that prolactin (PRL) might well be one of these factors. This study was designed to determine the effect of hypophysectomy on biochemical markers of exorbital lacrimal gland secretory capacity and to determine the extent to which dihydrotestosterone (DHT) and prolactin reverse these changes. METHODS: Female rats were hypophysectomized and, 5 days later, were treated for 2 days with DHT (0.25 or 1 mg/kg), PRL (1 or 5 mg/kg), combinations of the low or high doses of DHT and PRL, or vehicle only. The animals were killed, and crude membrane fractions were isolated from their lacrimal glands. An untreated group served as control. RESULTS: Lacrimal glands atrophied rapidly after hypophysectomy, losing 40% of their total and membrane-associated protein and 50% of their total DNA within 5 days. Total Na+,K(+)-ATPase and acid phosphatase activities and beta-adrenergic receptor number were decreased by half, whereas alkaline phosphatase activity and muscarinic cholinergic receptor number were reduced by 25% to 30%. DHT treatment increased total DNA above control values; it partially restored the amount of protein in the gland, the Na+,K(+)-ATPase and acid phosphatase activities, and the beta-adrenergic receptor number; and it fully restored the alkaline phosphatase activity. Prolactin treatment partially restored the amount of protein in the gland and the Na+,K(+)-ATPase activity; it fully restored the alkaline phosphatase activity and cholinergic receptor number; but it had no effect on the acid phosphatase activity or the beta-adrenergic receptor number. The high dose of DHT reduced the increase in cholinergic receptor number elicited by PRL. The high dose of PRL reduced the increases of total Na+,K(+)-ATPase and acid phosphatase elicited by DHT. CONCLUSIONS: These findings suggest that DHT and PRL exert general trophic actions on the lacrimal gland and specifically on lacrimal Na+,K(+)-ATPase, acid phosphatase, and neurotransmitter receptors. They also suggest that excessive levels of either hormone may be deleterious to secretory function. Because sex hormone levels are prone to wide fluctuations in women, our results also suggest a plausible hypothesis to account for the greater incidence in women of lacrimal insufficiency.

Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration.

PURPOSE: Previous studies have shown that ovariectomy and hypophysectomy cause regression of the lacrimal gland and have implicated androgens as trophic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apoptosis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-induced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS: Groups of sexually mature female New Zealand White rabbits were ovariectomized and killed at various time periods up to 9 days. Additional groups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At each time period, sham-operated rabbits were used as controls. Lacrimal glands were removed and processed for analysis of apoptosis as assessed by DNA fragmentation and for morphologic examination. DNA fragmentation was determined using the TdT-dUTP terminal nick-end labeling assay and by agarose gel electrophoresis. Labeled nuclei were quantified by automated densitometry. Sections were also stained for RTLA (rabbit thymic lymphocyte antigen), rabbit CD18, and La antigen. Morphology was evaluated by both light and electron microscopy. RESULTS: The time course of apoptosis exhibited two phases, a rapid and transient phase and a second prolonged phase. A transient phase peaked at approximately 4 to 6 hours after ovariectomy. The values for degraded DNA as a percentage of total nuclear area were 4.29%+/-0.79% and 4.26%+/-0.54%, respectively. The values for sham-operated controls examined at the same time periods were 1.77%+/-0.08% and 0.82%+/-0.21%, respectively. The percentage of degraded DNA at 24 hours after ovariectomy was not different from controls examined at the same interval after sham operation. The percentage of degraded DNA 6 days after ovariectomy was significantly increased (8.5%+/-2.4%), compared with sham-operated animals at the same time period (0.68%+/-0.03%). DNA laddering was more pronounced after ovariectomy. Dihydrotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections indicated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ovariectomy showed nuclear chromatin condensation principally in plasma cells. Increased numbers of macrophages were also evident. Significant levels of cell degeneration and cell debris, characteristic of necrosis, were observed in acinar regions 6 days after ovariectomy. Dihydrotestosterone prevented this necrosis. Increased numbers of RTLA+, CD18+, and La+ interstitial cells were also evident 6 days after ovariectomy. In addition, ovariectomy increased La expression in ductal cells. Dihydrotestosterone treatment prevented the increase in numbers of lymphoid cells and La expression. Dihydrotestosterone also promoted the appearance of mitotic figures in acinar cells and increased the sizes of acini by 43% (P < 0.05). CONCLUSIONS: Glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosis. These processes are accompanied by increased lymphocytic infiltration. These results suggest that a critical level of androgen is necessary to maintain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necrosis, and an autoimmune response. Because apoptotic and necrotic cell fragments may be sources of autoantigens that can be processed and presented to initiate an autoimmune reaction, we surmise that cell death triggered by androgen withdrawal may trigger an autoimmune response such as that encountered in Sjögren's syndrome. (ABSTRACT TRUNCATED)

Transport properties of intestinal basolateral membranes.

Techniques for the isolation and study of basolateral membrane vesicles from the intestinal epithelium have afforded new insights into the mechanisms of intestinal absorption. First, we have confirmed the hypothesis that the second stage of glucose transport involves facilitated diffusion. Second, we have shown that the major system for translocation of neutral amino acids across the basolateral membrane is the classical "L" system. Third, we have established that basolateral membranes contain sodium-dependent transport systems that may be useful in the supply of essential amino acids to the epithelium from the blood. And, finally, our studies of the basolateral (Na + K)-ATPase have clarified the role of this enzyme in sodium absorption.

Evidence of sodium-dependent glucose transport in human erythroleukemia cells.

Sodium-dependent transport of D-glucose has been reported only in epithelial cells of small intestine and kidney, and well-differentiated tumors thereof. We observed a two-fold decrease (p < 0.05) in the intracellular distribution volume (Vic, defined as steady-state intracellular uptake divided by extracellular concentration) of the non-metabolized D-glucose analog 3-O-methylglucose (3-O-MG) when logarithmically growing K562 cells (an anaplastic human erythroleukemia) were incubated 3 h in choline-substituted, phosphate buffered saline (PBS) rather than Na+ PBS, each containing a glucose concentration ([Glu]) of 5.6 mM. Electromechanically measured cellular volume Vc differed < 10% between the different media. In Na+ PBS, Vic (3-O-MG) was approximately twice Vc and declined progressively when [Glu] was reduced to 2.8 and 0.1 mM. We conclude that, in a balanced salt medium containing glucose as the only energy source, K562 cells express a concentrative mechanism having characteristics consistent with Na(+)-dependent transport of glucose.